Induced pluripotent stem cell-derived cardiomyocyte in vitro models: tissue fabrication protocols, assessment methods, and quantitative maturation metrics for benchmarking progress

The advent of human induced pluripotent stem cells (hiPSCs) and techniques to differentiate cardiomyocytes from them has opened a viable path to creating in vitro models of normal and diseased hearts, accelerating more predictive drug screening and therapeutic strategies for cardiac pathologies. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) are more similar to fetal than adult cardiomyocytes, leading many in the field to explore approaches to enhance cell and tissue maturation. There are over 2,000 studies utilizing hiPSC-CMs in models composed of various combinations of cell and extracellular matrix components, using a plethora of differentiation protocols, culture formats, and methods for quantifying cardiomyocyte function. To assess the current state of this rapidly growing area, we systematically analyzed 300 studies using hiPSC-CM models for their selection of hiPSC lines, hiPSC-CM differentiation protocols, types of in vitro models, maturation techniques, and metrics used to assess cardiomyocyte functionality and maturity. Here, we provide the data compiled from our analysis of these papers so others in the field can utilize it to inform their research. Based on this analysis, we highlight the diversity of, and current trends in, in vitro model designs and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology, and gene expression and noted field-wide improvements over time. Finally, we observe that a persistent lack of coordination amongst investigators is limiting the field’s ability to benchmark and advance hiPSC-CM function against previous studies. We discuss opportunities to collectively pursue the common goal of hiPSC-CM model development, maturation, and assessment that we believe are critical to driving the entire community forward in engineering mature cardiac tissue.

Methods
Across over 2,000 studies utilizing hiPSC-CMs from 2016-2022, there exists high variability in differentiation protocols, maturation strategies, and assessment of cell or tissue functionality. To understand the nature of this variability, we systematically analyzed 300 studies using hiPSC-CM models and compiled data on hiPSC lines, hiPSC-CM differentiation protocols, types of 2D and 3D in vitro models, maturation techniques, and metrics used to assess hiPSC-CM functionality and maturity. Using a PubMed search for “(((induced pluripotent stem cells) OR (iPSC)) AND ((cardiomyocyte) OR (cardiomyocytes) OR (iPSC-CM) OR (iPSC-CMs) OR (induced pluripotent stem cell-cardiomyocytes) OR (induced pluripotent stem cell-CMs))) AND ((engineered heart tissue) OR (cardiac microtissue) OR (maturation) OR (mature))” we identified 846 potential publications for analysis. We eliminated publications focusing on atrial differentiation due to relatively limited published work in this space and focused our benchmarking on ventricular cardiomyocytes, which are of particular interest in the development of cardiac regenerative therapies. We also eliminated publications that were specifically focused on disease modeling or mechanistic studies unless maturation techniques (the primary focus of our analysis) were utilized in the study. To capture overall trends in the field, we focused on 300 publications. This included the top 100 cited publications from the 846 potential publications based on citation numbers reported by the Web of Science database as of 2022, 100 publications from the PubMed 846 potential publications (that were not the 100 top-cited publications) selected randomly, and 100 additional hiPSC-CM publications that were manually selected from references in recent high impact reviews on cardiac tissue engineering or top search results for “iPSC-cardiomyocytes” on Google Scholar that utilized unique culture platforms, maturation techniques, and/or assessments.